Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00
Description: Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

We sincerely thank Dr. Mustafa Khokha, Yale University, for providing the use of his laboratory resources. We are grateful to Mr. Ming-Jie Wu for excellent technical assistance. EF is an HHMI investigator.This work was supported by a gift from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund.

1. Preparation of DNA Templates
<ol>
	<li>Download the 16S rRNA sequence specific to each bacterial genera from the NCBI database and design polymerase chain reaction (PCR) compatible forward and reverse primers that will amplify genera-specific regions (~200 - 250 base-pair long) as described earlier19. Also refer to the open source databases SILVA20,21 and probeBase22 online resources for rRNA-targeted oligonucl...

<ol>
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This is the first use of a whole-mount in situ hybridization (WMISH) technique to study the microbiota of an arthropod vector of pathogens. Our protocol was adapted from one used to study development in Drosophila and in frog embryos25,26. Whole-mount RNA in situ hybridization has been routinely used to localize gene transcripts spatially and temporally27 and visualization of transcripts can be done by bright-fie...

Human and livestock pathogens transmitted by arthropod vectors are found worldwide and account for about 20% of infectious diseases globally1, but effective and safe vaccines against most of these pathogens are not available. Our understanding of the important role of commensal, symbiotic and pathogenic microorganisms, collectively known as the microbiome2, in modulating and shaping the health of almost all metazoans3 is expanding. It is now evident that arthropod vectors of pathogens also harbor gut microbiota and these vector-associated microbiota have been shown to influence diverse vector-borne pathogens4,5. The arthropod microbiome is composed of eubacteria, archaea, viruses, and eukaryotic microbes such as protozoa, nematodes, and fungi6. However, the predominant research focus has been on eubacteria due, in part, to the availability of marker genes and reference databases to identify specific bacterial members.
With a focus on Ixodes scapularis, the tick vector of multiple human pathogens including Borrelia burgdorferi7, the causative agent of Lyme disease, the optimization of a microbial visualization technique was aimed at improving our understanding of tick gut microbiota in the context of vector-pathogen interactions. Several questions remain to be answered in the tick microbiome field. The gut is the site of the first extended encounter between the tick and the incoming pathogen in the context of horizontally transferred pathogens; therefore, understanding the role of vector gut microbiota in modulating vector-pathogen interactions will reveal meaningful insights. Ticks have a unique mode of blood meal digestion, where processing of blood meal components takes place intracellularly8. The gut lumen seemingly serves as a vessel to contain the blood meal as the tick feeds, and nutrient digestion and assimilation ensue throughout the several days of feeding and continue post-repletion. The pathogens acquired by the tick during feeding enter the gut lumen along with the bloodmeal and thus the lumen becomes a primary site of interactions among the tick, pathogen, and resident microbiota. As digestion proceeds through the repletion and Ixodid tick molting, the gut undergoes structural and functional changes9. The composition and the spatial organization of gut bacteria is also likely to vary in concert with the changing gut milieu. It is, therefore, important to understand the architecture of resident bacteria in the tick gut to fully understand the interplay of tick, pathogen, and gut microbiota.
Molecular techniques to describe host-associated microbiota routinely utilize high-throughput parallel sequencing strategies10 to amplify and sequence bacterial 16S ribosomal DNA (rDNA). These sequencing strategies circumvent the need to obtain axenic cultures of specific bacteria and provide an in-depth description of all bacterial members represented in the sample. Nevertheless, such strategies are confounded by the inability to distinguish environmental contaminations from bona fide residents. Further, when assessing samples, such as ticks, that are small in size and hence contain low microbiota-specific DNA yields, the likelihood of amplification of environmental contaminants is increased11 and results in the ambiguous interpretation of microbiome composition. Functional characterization in conjunction with the visualization of specific viable bacteria will, therefore, be critical to define and discern the microbiome of the tick temporally and spatially. Towards this goal, we took advantage of the whole-mount RNA in situ hybridization. This technique is routinely used to assess gene expression patterns in organs and embryos12,13,14 and allows semiquantitative analysis of expression over the entire sample of interest. This differs from traditional in situ hybridization techniques which utilize tissue sections and often require extensive analysis of sectioned material with a computational assembly to predict expression in whole organs15. While whole-mount generally refers to whole organisms12, here whole-mount refers to whole guts or organs. The advantages of using the whole-mount RNA in situ hybridization approach to assess the architecture of tick gut microbiota are multifold. The tick gut is composed of 7 pairs of diverticula, each pair varying in size16. The functional differences, if any, among these diverticula, are not understood in the context of tick biology, tick microbiota or tick-pathogen interactions. Manipulations of the gut that rupture the gut diverticula would displace microbiota present in the gut lumen or those associated loosely with the gut and result in misinterpretation of the spatial localization of microbiota. Fluorescence-labeled RNA in situ hybridization has been utilized earlier to examine tick gut transcripts17 by fixing and opening individual gut diverticula to ensure probe hybridization and to localize B. burgdorferi transcripts by sectioning paraffin-embedded whole ticks18. Both these approaches require manipulations of the tick tissues prior to hybridization that would affect the gut microbiota architecture.
In this report, we describe in detail the protocol to examine viable tick gut microbiota using whole-mount in situ hybridization (WMISH). The use of whole-mount RNA in situ hybridization enables a global understanding of the presence and abundance of specific gut bacteria in the different regions of the gut and may spur new insights into tick gut biology in the context of pathogen colonization and transmission. Further, the use of RNA probes directed against specific bacterial RNA allows detection of viable bacteria in the tick gut.

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			<td height="21" style="height:21px;width:320px;">pGEM-T Easy Vector System</td>
			<td style="width:220px;">Promega</td>
			<td style="width:348px;">A1360</td>
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			<td height="21" style="height:21px;width:320px;">Digoxygenin-11-UTP</td>
			<td style="width:220px;">Roche</td>
			<td align="right" style="width:348px;">1209256910</td>
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			<td height="21" style="height:21px;width:320px;">dNTP</td>
			<td style="width:220px;">New England Biolabs&#160;</td>
			<td style="width:348px;">N0447S</td>
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			<td height="21" style="height:21px;width:320px;">DNAse I(RNAse-free)</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe SP6 RNA synthesis kit</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe T7 High Yield RNA Synthesis Kit</td>
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			<td height="21" style="height:21px;width:320px;">Water, RNase-free, DEPC-treated</td>
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			<td style="width:348px;">AB02128-00500</td>
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			<td height="21" style="height:21px;width:320px;">EDTA, 0.5M, pH 8.0</td>
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			<td height="21" style="height:21px;width:320px;">Formaldehyde, 37%</td>
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			<td height="21" style="height:21px;width:320px;">Formamide</td>
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			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">E-4378</td>
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			<td height="21" style="height:21px;width:320px;">RNA from torula yeast</td>
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			<td height="21" style="height:21px;width:320px;">Heparin, sodium salt</td>
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			<td height="21" style="height:21px;width:320px;">Denhardt&#39;s Solution, 50X</td>
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			<td height="21" style="height:21px;width:320px;">RNase A</td>
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			<td style="width:348px;">STEMI 2000-C</td>
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			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">Zen lite</td>
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			<td height="21" style="height:21px;width:320px;">Image editing software&#160;</td>
			<td style="width:220px;">Adobe&#160;</td>
			<td style="width:348px;">Adobe Photoshop CS4 version 11.0</td>
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			<td height="21" style="height:21px;width:320px;">Image analysis software</td>
			<td style="width:220px;">National Institutes of Health</td>
			<td>ImageJ-NIH /imagej.nih.gov/ij/</td>
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		</tr>
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			<td height="64" style="height:64px;">Automation compatible instrumentation</td>
			<td style="width:220px;">Intavis Bioanalytical Instruments, Tubingen, Germany).&#160;</td>
			<td style="width:348px;">Intavis, Biolane HT1.16v</td>
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		</tr>
	</tbody>
</table>

visualization of microbiota in tick guts by whole-mount in situ hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

The measurement and estimation of the quality of the RNA probes are critical prior to beginning the staining. In vitro transcription efficiency depends highly on the amount and quality of the DNA template. We routinely visualized the RNA probes on a formaldehyde gel to verify the purity and amount of probe generated by the transcription reactions. The probes should appear as bright, discrete bands (Figure 2). Spectrophotometric measurements of RNA co...

Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Research

JoVE Journal

Genetics

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